Borrelia burgdorferi, the causative agent of Lyme disease, is a tick-borne bacterial pathogen that must adapt to varying environments in its tick and mammalian hosts. B. burgdorferi has been shown to significantly alter protein expression to adapt to environmental changes, but factors that trigger these changes and the mechanisms behind them are still not well understood. The availability of carbon sources for use by the bacteria differ substantially in mammals, where glucose is the primary carbon source available, and in ticks, where glycogen, trehalose, and chitobiose may be more readily available. For other bacteria, changes in the carbon source being utilized is important in regulating expression of genes involved in carbon metabolism, as well as virulence factors, toxins and other factors important in adapting to specific environments. Whether B. burgdorferi utilizes carbon source availability to adapt to environments is not known; however several B. burgdorferi genes related to carbon utilization have been shown to be critical for survival in specific hosts. Studies to understand the genetic determinants of environmental adaptation by B. burgdorferi have been slowed by a paucity of genetic tools-particularly those amenable to high-throughput screening. Massively parallel sequencing is a rapidly developing technology for the study of bacterial pathogenesis. Dr. Andrew Camilli, a co-investigator on this proposal, has paired massively parallel sequencing with transposon mutagenesis in a strategy called Tn-seq. Tn-seq involves screening of a library against a selective pressure and then sequencing the flanking regions to the transposon en masse, to identify the relative frequency of the mutants before and after selection. Dr. Tao Lin's laboratory has developed the first transposon mutant library in B. burgdorferi. In our preliminary studies, we have successfully employed Tn-seq on B. burgdorferi transposon mutants grown in vitro and in vivo in mice and shown that it is a sensitive and reproducible technique. The screening of subsets of the library in mice revealed that several of the genes likely to be involved in carbon utilization show either increased or decreased fitness for survival in mice. In this proposal, we examine the role of carbon utilization by B. burgdorfer. We will screen our transposon library by Tn-seq after growth in the presence or absence of specific sugars. Based on analysis of this data, which we expect will identify genes involved in uptake and metabolism of specific carbon sources as well as global regulators involved in controlling genetic programs for environmental adaptation, we will select a subset of 5 genes for testing by creation of targeted deletion mutants and complemented mutants. These strains will be tested for survivial in ticks and mice and in competition assays. The development of Tn-seq for application to studies of B. burgdorferi has the potential to greatly accelerate our understanding of the pathogenesis of this organism and the mechanisms by which it adapts to its hosts.